Heterologous Gene Expression System Using the Cold-Inducible CnAFP Promoter in Chlamydomonas reinhardtii

Abstract

To increase the availability of microalgae as producers of valuable compounds, it is necessary to develop novel systems for gene expression regulation. Among the diverse expression systems available in microalgae, none are designed to induce expression by low temperature. In this study, we explored a cold-inducible system using the antifreeze protein (AFP) promoter from a polar diatom, Chaetoceros neogracile. A vector containing the CnAFP promoter (pCnAFP) was generated to regulate nuclear gene expression, and reporter genes (Gaussia luciferase (GLuc) and mVenus fluorescent protein (mVenus)) were successfully expressed in the model microalga, Chlamydomonas reinhardtii. In particular, under the control of pCnAFP, the expression of these genes was increased at low temperature, unlike pAR1, a promoter that is widely used for gene expression in C. reinhardtii. Promoter truncation assays showed that cold inducibility was still present even when pCnAFP was shortened to 600 bp, indicating the presence of a low-temperature response element between -600 and -477 bp. Our results show the availability of new heterologous gene expression systems with cold-inducible promoters and the possibility to find novel low-temperature response factors in microalgae. Through further improvement, this cold-inducible promoter could be used to develop more efficient expression tools.

Keywords: Chlamydomonas reinhardtii; Cold-inducible expression system; Gaussia luciferase; antifreeze protein; fluorescent protein; truncated promoter.

Fig. 1. Generation of the pCnAFP vector construct and verification of transformants.

(A) Vector backbone pChlamy3_Empty_GLuc (left) does not have a promoter sequence in front of the Gaussia luciferase gene (GLuc). Vector backbone pCnAFP_GLuc (right) with the 1,225 bp pCnAFP insertion. (B and C) Confirmation of vector insertion in transformants by colony PCR (B, hygromycin resistance gene, 467 bp; C, region connecting pCnAFP and GLuc, 964 bp). Lanes 1: plasmid positive for pCnAFP_GLuc, lanes 2: wild-type C. reinhardtii, lanes 3–5: pCnAFP_GLuc transformants.

Fig. 2. Inducibility test of pCnAFP_GLuc transformants.

(A) Relative GLuc mRNA levels after 2 h treatment at the low-temperature conditions; (B) Relative luminescence levels after 12 h treatment at the low-temperature conditions; (C) Relative GLuc mRNA levels at 10°C; (D) Relative luminescence levels at 10°C. (A) and (B) were presented with the relative value calculated by the value at the optimal culture temperature of C. reinhardtii (25°C). All experiments were conducted with at least four replicates. Statistical analyses were performed using the Student’s t-test, *p < 0.05.

Fig. 3. Comparison of the strength and cold inducibility of pCnAFP and pAR1.

(A) Promoter strength of pCnAFP and pAR1 based on the relative mRNA expression of GLuc at 25°C. The expression level of pCnAFP was calculated to relative value based on that of pAR1; (B) Relative levels of GLuc mRNA expression driven by pCnAFP and pAR1 at 10°C. In (B), cold inducibility was calculated by comparing to each value in 25°C of pAR1 and pCnAFP. All experiments were conducted in more than triplicate. Statistical analyses were performed using the Student’s t-test, *p < 0.05.

Fig. 4. Cold-inducibility test of transformants carrying progressively truncated pCnAFP_GLuc variants.

(A) Schematic representation of truncated promoters; (B) Relative levels of GLuc expression driven by truncated pCnAFPs in response to exposure to 10°C. Cold inducibility was confirmed by calculations based on the value of 25°C. All experiments were conducted with at least four replicates. Statistical analyses were performed using the Student’s t-test, *p < 0.05.

Fig. 5. Expression of pCnAFP_mVenus in C. reinhardtii under low-temperature conditions.

(A) Vector backbone of pChlamy3_Empty_mVenus (left) does not have a promoter sequence in front of the mVenus fluorescent protein gene (mVenus). Vector backbone of pCnAFP_mVenus (right) with either 1,225 bp pCnAFP or 600 bp pCnAFP. (B and C) Confirmation of vector insertion in transformants by colony PCR with two primer sets (B, 467 bp for hygromycin resistance gene; C, 1,519 bp for the region connecting pCnAFP and mVenus). Lanes 1: plasmid positive for pCnAFP 1,225 bp_mVenus, lanes 2: plasmid positive for pCnAFP 600 bp_mVenus, lanes 3: wild-type C. reinhardtii, lanes 4–6: pCnAFP 1,225 bp_mVenus transformants, lanes 7–9: pCnAFP 600 bp_mVenus transformants. (D) Changes in the relative levels of mVenus mRNA in response to low temperature (10°C). Cold inducibility was confirmed by calculating mRNA levels, which are relative to those at 25°C. All experiments were conducted in more than triplicate. (E) Fluorescent images of transformants after low-temperature treatment (0h, 4h, and 8h). Merged images of mVenus (emission at 537–559 nm, excitation at 502–522 nm) and chlorophyll fluorescence (emission at 603–648 nm, excitation at 563–588 nm) are shown.